Adeno-associated virus (AAV) is a defective parvovirus whose genome is encapsidated as a single-stranded DNA molecule. Strands of plus and minus polarity are packaged with equal efficiency, but in separate virus particles. Efficient replication of AAV generally requires coinfection with a helper virus of the herpesvirus or adenovirus family, although under special circumstances, AAV can replicate in the absence of helper virus.
In the absence of helper virus, AAV establishes a latent infection in which the viral genome exists as an integrated provirus in the host cell. Integration of the virus occurs on human chromosome 19. If a latently infected cell line later is superinfected with a suitable helper virus, generally the AAV provirus is excised and the AAV virus enters the xe2x80x9cproductivexe2x80x9d phase.
AAV isolates have been obtained from human and simians. The host range for lytic growth of AAV is broad. Cell lines from virtually every mammalian species tested, including a variety of human, simian, canine, bovine and rodent cell lines, can be infected productively with AAV, provided an appropriate helper virus is used (e.g., canine adenovirus in canine cells) . However, no disease has been associated with AAV in either human or other animal populations.
AAV has been isolated as a nonpathogenic coinfecting agent from fecal, ocular and respiratory specimens during acute adenovirus infection, but not during other illnesses. Latent AAV infections have been identified in both human and nonhuman cells. Overall, virus integration appears to have no apparent effect on cell growth or morphology, see Samulski, Curr. Op. Gen. Devel., 3:74-80 (1993).
There are a number of AAV""s, including AAV-1, AAV-2, AAV-3, AAV-4 and AAV-5. The genome of AAV-2 is 4,679 bases in length (Genbank No. AF043303) and contains inverted terminal repeat sequences of 145 bases each. The repeats are believed to act as origins of DNA replication. The AAV-3 genome is 4726 bases in length and has 82% overall sequence homology with AAV-2, see Muramatsu, Virology, 221:208-217 (1996). Like AAV-2, both ends of the AAV-3 genome consist of inverted repeats but the palindromes are 146 bp in size. Certain portions of the AAV-2 and AAV-3 genomes are highly conserved, for example, there are two sites in the hairpin where there is only a single base pair substitution between AAV-2 and AAV-3.
The AAV genome has two major open reading frames. The left frame encodes at least four non-structural proteins (the Rep group). There are two promoters, P5 and P19, which control expression of those proteins. As a result of differential splicing, the P5 promoter directs production of proteins Rep 78 and Rep 68, and the P19 promoter, of the proteins Rep 52 and Rep 40. The Rep proteins are believed to be involved in viral DNA replication, trans-activation of transcription from the viral promoters, repression of heterologous enhancers and promoters as well as with site-specific integration.
The right ORF, controlled by the P40 promoter, encodes the capsid proteins, Vp1 (91 kDa), Vp2 (72 kDa) and Vp3 (60 kDa). Vp3 comprises 80% of the virion structure while Vp1 and Vp2 are minor components. There is a polyadenylation site at map unit 95. For the complete sequence of the AAV-2 genome, see Strivastava et al., J. Virol., 45:555-64 (1983).
McLaughlin et al., J. Virol., 62:1963-73 (1988) prepared two AAV vectors: dl 52-91, which retains the AAV rep genes, and dl 3-94 in which all of the AAV coding sequences are deleted. dl 3-94 does, however, retain the two 145 base terminal repeats and an additional 139 bases which contain the AAV polyadenylation signal. A foreign gene, encoding neomycin resistance, was inserted into the vector. Viral stocks were prepared by complementation with a recombinant AAV genome which supplied the missing AAV gene products in trans but was itself too large to be packaged. Unfortunately, the virus stocks were contaminated with wild type AAV (10% in the case of dl 3-94) presumably as a result of homologous recombination between the defective and the complementing viruses.
Samulski et al., J. Virol., 63:3822-28 (1989) developed a method of producing recombinant AAV stocks without detectable wild-type helper AAV. The AAV vector retained only the terminal 191 bases of the AAV chromosome. In the AAV helper plasmid (pAAV/Ad), the terminal 191 bases of the AAV chromosome were replaced with adenovirus terminal sequences. Since sequence homology between the vector and the helper AAV thus essentially was eliminated, no detectable wild-type AAV was generated by homologous recombination. Moreover, the helper DNA itself was not replicated and encapsidated because the AAV termini are required for that process. Thus, in the AAV system, unlike the HSV system, helper virus could be eliminated completely leaving a helper-free AAV vector stock.
Recombinant AAV (rAAV) vectors have been used for expressing gene products in animals, see, for example, U.S. Pat. No. 5,193,941 and WO 94/13788. Other patents and publications describe AAV vectors and uses, the uses generally being related to expression of gene products either in vitro (usually tissue cultures) or in vivo (usually in the lungs or oral mucosa, the normal sites of AAV infection, but expression in other tissues, such as the central nervous system and in cardiac tissue has been observed).
Transduction of rAAV vectors harboring the bacterial xcex2-galactosidase gene by single injection into the quadriceps of mice demonstrated that expression was maintained long-term and the expression did not decrease substantially during that time (Xiao et al., J. Virol., 70:8098-8108 (1996)). Other targets successfully transduced with rAAV vectors include: T-lymphocytes and B-lymphocytes, human erythroleukemia cells, different regions of the rat brain, the striatum of the rat brain in a Parkinson""s Disease model with the tyrosine hydroxylase gene, heart of the pig and rat with the LacZ gene, the peripheral auditory system of the guinea pig and bronchial epithelia of the rabbit and monkey. In addition, a Phase I human clinical trial for the delivery of an rAAV-CFTR vector is in progress.
AAV""s harboring the human factor IX gene were infused into the portal vein of adult immunocompetent mice and long-term gene expression was obtained (Snyder et al., Nat. Genet., 16:270-276 (1997)). The vectors were found to be integrated into the murine genome (Miao et al., Nat. Genet., 19:13-15 (1998)).
Hemophilia A is an X chromosome-linked bleeding disorder resulting from a deficiency of or an abnormality of factor VIII (FVIII or fVIII), a component of the coagulation cascade. The human FVIII cDNA has been cloned. FVIII is synthesized as a 2351 amino acid residue, single chain precursor composed of a 19 amino acid signal peptide and six distinct domains. The domains are arranged in the order, A1-A2-B-A3-C1-C2 (Toole et al., Nature, 312:342-347 (1984); Vehar et al., Nature, 312:337-342 (1984)). An A domain contains about 330 amino acids and is present in three copies. A C domain contains about 150 amino acids and is present in two copies. The B domain contains about 909 amino acids and is extremely rich in potential N-linked glycosylation sites.
The translation product of the FVIII gene first is cleaved between the B domain and the A3 domain. Then, the B domain is proteolysed at multiple sites leaving FVIII as a divalent metal ion-linked complex consisting of the heavy chain (H chain) of 90-200 kDa and the light chain (L chain) of 80 kDa (Vehar et al., (1984), supra; Anderson et al., Proc. Natl. Acad. Sci. USA, 83:2979-2983 (1986)).
The minimal functional unit of FVIII is the heterodimer consisting of the 90 kDa H chain and the 80 kDa L chain. Thus, the B domain is dispensable for procoagulant activity (Eaton et al., Biochemistry, 25:8343-8347 (1986); Toole et al., Proc. Natl. Acad. Sci., USA 83:5939-5942 (1986)). Circulating FVIII in blood is associated with the von Willebrand factor (vWF) which is a large multimeric, multifunctional product (Brinkhous et al., Proc. Natl. Acad. Sci. USA, 82:8752-8756 (1985)).
Hemophilia A patients are at risk of contracting transmissible infectious diseases from plasma-derived FVIII used in treatment. Thus, recombinant product is a desirable alternative. However, the complicated processing and large size of FVIII have hampered production of FVIII in prokaryotes or lower eukaryotes.
Expression of full-length FVIII cDNA in mammalian cells was reported by several groups, but the levels of expression were very low and insufficient for economical production of recombinant FVIII (rFVIII).
To improve expression efficiency, modified FVIII cDNA""s lacking most of the B domain were made (Eaton et al., (1986), supra; Toole et al., (1986), supra; Sarver et al., Behring Inst. Mitt., 82:16-25 (1988); Meulien et al., Protein Engng., 2:301-306 (1988); Tajima et al., Proc. 6th Int. Symp., II.T:51-63 (1990)) and the resulting products were shown to retain functional activities of FVIII.
Tajima et al., (1990), supra, fused the coding sequences of the H and L chains. Although that construct was expressed about 10-fold higher than a full length FVIII cDNA construct, 20% of the product was not cleaved to the H and L chains or was cleaved incorrectly. Eaton et al. ((1986), supra) inserted a junction peptide derived from the B-domain between the H and L chains. However, the junction peptide remained at the C terminus of the H chain. Such fusion molecules have antigenic properties (Esmon et al., Blood, 76:1593-1600 (1990)) which can elicit serious side effects because of the constant exposure of the host to those antigens during the extended duration of treatment.
Burke et al. (J. Biol. Chem. 261:12574-12578 (1986)) expressed the H chain (Ala1-Arg740) and the L chain (Glul649-Tyr2332) as separate proteins in COS cells and observed secretion of functionally active FVIII. But the expression levels were even lower than that of the full length construct. Yonemura et al. (Prot. Engng., 6:669-674 (1993) essentially duplicated those efforts using plasmids in CHO cells.
Another complication of the disease is the observation that the severity of the bleeding tendency varies among patients and may be related to the concentration of functional clotting factors. Individuals can have mild hemophilia that may not be recognized until adulthood or following heavy trauma or surgery, see, for example, Reiner and Davie, xe2x80x9cIntroduction to hemostasis and the vitamin K-dependent coagulation factorsxe2x80x9d in The Metabolic Basis of Inherited Disease (Scriver et al., eds.) Vol. 3, pages 3181-3221 (McGraw Hill, N.Y., 1995).
Adenovirus vectors can infect non-dividing cells and therefore, can be delivered directly into mature tissue, such as muscle. However, the transgenes delivered by adenovirus vectors are not useful for long term expression for a variety of reasons. First, adenovirus vectors retain most of the viral genes and thus pose potential problems, i.e. safety. Expression of the adenovirus genes can cause the immune system to destroy the cells containing the vectors (see, for example, Yang et al., Proc. Natl. Acad. Sci. 91:4407-4411 (1994)). Since adenovirus is not an integration virus, the vector eventually will be diluted or degraded in the cells. Also, because of the immune response, adenovirus vectors cannot be delivered repeatedly. In the case of lifetime disease, such as the hemophilias, that will be a major limitation.
For retrovirus vectors, although stable integration into the host chromosomes can be achieved, the use thereof is restricted because currently used vectors only can infect dividing cells, a large majority of target cells being non-dividing.
AAV vectors have certain advantages over the above-mentioned vector systems. First, like adenovirus, AAV infects non-dividing cells. Second, all the AAV viral genes are eliminated in the vector. Since the viral gene expression-induced immune reaction is no longer a concern, AAV vectors are safer than adenovirus vectors. As AAV is an integration virus, integration into the host chromosome will maintain the transgene in the cells. AAV is an extremely stable virus, resistant to many detergents, pH changes and heat (stable at 56xc2x0 C. for about an hour). AAV can be lyophilized and redissolved without losing significant activity. Finally, AAV causes no known diseases or pathogenic symptoms in humans. Therefore, AAV is a very promising delivery vehicle for gene therapy.
Two recent review articles provide an overview of the recent status on the use of AAV vectors and include a collection of additional recent scientific publications in the field: Samulski, xe2x80x9cAdeno-associated Viral Vectorsxe2x80x9d, Chap. 3 in xe2x80x9cViruses in Human Gene Therapyxe2x80x9d, Vos et al., ed., Chapman and Hall, 1994; and Samulski, xe2x80x9cAdeno-associated Virus-based Vectors for Human Gene Therapyxe2x80x9d, Chap. 11 in xe2x80x9cGene Therapy: From Laboratory to the Clinicxe2x80x9d, Hui et al., ed., World Scientific, 1994.
Since AAV has in the past been shown to have a broad host range; can be administered by a variety of routes, including intramuscular injection; and is operable in different cells types, such as liver, retina, neurons and so on, there are no known limits of the host in which the herein described methods of delivery can take place, particularly in, for example, mammals and birds, especially domesticated mammals and birds, such as cattle, sheep, pigs, horses, dogs, cats, chickens and turkeys. Both human and veterinary uses particularly are preferred.
The instant invention provides recombinant AAV vectors for effective expression of a protein with Factor VIII function to treat hemophilia A and homologous disorders. The recombinant AAV vectors described herein provide a significant development in the field of recombinant AAV vector gene therapy.
The rAAV vectors of the invention can be used as viral particles alone. Alternatively, the rAAV vector virus particles can be used in conjunction with additional treatments, including partial hepatectomy or treatment with secondary agents that enhance transduction, whether associated with in vivo or ex vivo therapies. Examples of secondary agents include gamma irradiation, UV irradiation, tritiated nucleotides such as thymidine, cis-platinum, etoposide, hydroxyurea, aphidicolin and adenovirus.